PD-1 peptide inhibitors

ABSTRACT

This disclosure provides peptides which have a strong affinity for the checkpoint receptor “programmed death 1” (PD-1). These peptides block the interaction of PD-1 with its ligand PD-L1 and can therefore be used for various therapeutic purposes, such as inhibiting the progression of a hyperproliferative disorder, including cancer, treating infectious diseases, enhancing a response to vaccination, and treating sepsis.

This application is a divisional application of Ser. No. 15/705,333filed on Sep. 15, 2017, which claims priority to and incorporates byreference in its entirety U.S. Ser. No. 62/395,195 filed on Sep. 15,2016. Each reference cited in this disclosure is incorporated herein inits entirety.

This application incorporates by reference the contents of a 1.43 kbtext file created on Feb. 27, 2018 and named “00047900251sequencelisting.txt,” which is the sequence listing for thisapplication.

TECHNICAL FIELD

This disclosure relates generally to immunomodulatory peptides.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Graph showing saturatable binding of anti-human PD-1 antibody toJurkat cells.

FIG. 2. Graph showing saturatable binding of PD-L1 Fc to Jurkat cells.

FIGS. 3A-B. Graphs showing effect of peptide QP20 on binding of PD-L1 toPD-1.

FIG. 3A, MFI; FIG. 3B, normalized mean fluorescence intensity (MFI).

FIGS. 4A-B. Graphs showing effect of peptide HD20 on binding of PD-L1 toPD-1.

FIG. 4A, MFI; FIG. 4B, normalized MFI.

FIGS. 5A-B. Graphs showing effect of peptide WQ20 on binding of PD-L1 toPD-1.

FIG. 5A, MFI; FIG. 5B, normalized MFI.

FIGS. 6A-B. Graphs showing effect of peptide SQ20 on binding of PD-L1 toPD-1.

FIG. 6A, MFI; FIG. 6B, normalized MFI.

FIG. 7A. Graph showing the effect of an anti-human PD-1 antibody on theinteraction between PD-1-expressing Jurkat T cells and PD-L1-expressingCHO cells that results in inhibition of a PD-1 mediated suppression ofluciferase reporter that is under the control of promoter containingIL-2, NFAT, and NF-kB response elements.

FIG. 7B. Graph showing the effect of an anti-human PD-1 antibody on theinteraction between PD-1-expressing Jurkat T cells and PD-L-expressingCHO cells (data in 7A expressed as fold inhibition).

FIG. 8A. Graph showing that PD-1 peptide inhibitors inhibit, in adose-dependent manner, the interaction between PD-1-expressing Jurkat Tcells and PD-L1-expressing CHO cells, which results in increasedluciferase reporter expression.

FIG. 8B. Graph showing the effect of an anti-human PD-1 antibody on theinteraction between PD-1-expressing Jurkat T cells and PD-L1-expressingCHO cells (data in 8B expressed as fold inhibition).

FIG. 9. Graph showing IL-2 production by peripheral blood mononuclearcells (PBMCs) in a tetanus toxoid recall assay after culture withpeptides QP20, HD20, WQ20, SQ20, or CQ-22.

FIG. 10. Graph showing IL-4 production by PBMCs in a tetanus toxoidrecall assay after culture with peptides QP20, HD20, WQ20, SQ20, orCQ-22.

FIG. 11. Graph showing IL-6 production by PBMCs in a tetanus toxoidrecall assay after culture with peptides QP20, HD20, WQ20, SQ20, orCQ-22.

FIG. 12. Graph showing IL-10 production by PBMCs in a tetanus toxoidrecall assay after culture with peptides QP20, HD20, WQ20, SQ20, orCQ-22.

FIG. 13. Graph showing IL-17a production by PBMCs in a tetanus toxoidrecall assay, after culture with peptides QP20, HD20, WQ20, SQ20, orCQ-22.

FIG. 14. Graph showing IFNγ production by PBMCs in a tetanus toxoidrecall assay, after culture with peptides QP20. HD20, WQ20, SQ20, orCQ-22.

FIG. 15. Graph showing TNFα production by PBMCs in a tetanus toxoidrecall assay, after culture with peptides QP20, HD20, WQ20, SQ20, orCQ-22.

FIG. 16. Graph showing IL-2 production by PBMCs in a tetanus toxoidrecall assay, after culture with various combinations of peptides QP20,HD20, WQ20, and SQ20.

FIG. 17. Graph showing IL-4 production by PBMCs in a tetanus toxoidrecall assay, after culture with various combinations of peptides QP20,HD20, WQ20, and SQ20.

FIG. 18. Graph showing IL-6 production by PBMCs in a tetanus toxoidrecall assay, after culture with various combinations of peptides QP20,HD20, WQ20, and SQ20.

FIG. 19. Graph showing IL-10 production by PBMCs in a tetanus toxoidrecall assay, after stimulation with various combinations of peptidesQP20, HD20, WQ20, and SQ20.

FIG. 20. Graph showing IL-17a production by PBMCs after stimulation withvarious combinations of peptides QP20, HD20, WQ20, and SQ20.

FIG. 21. Graph showing IFNγ production by PBMCs after culture withvarious combinations of peptides QP20, HD20, WQ20, and SQ20.

FIG. 22. Graph showing TNFα production by PBMCs after culture withvarious combinations of peptides QP20, HD20, WQ20, and SQ20.

FIG. 23A. Graph showing IL-2 production by PBMCs from donor A afterculture with peptides QP20, HD20, WQ20, and SQ20, or CQ-22.

FIG. 23B. Graph showing IL-2 production by PBMCs from donor B afterculture with peptides QP20, HD20, WQ20, or SQ20 and combinations ofthese peptides.

FIG. 24A. Graph showing IL-17a production by PBMCs from donor A afterculture with peptides QP20, HD20, WQ20, and SQ20, or CQ-22.

FIG. 24B. Graph showing IL-17a production by PBMCs from donor B afterculture with peptides QP20, HD20, WQ20, or SQ20 and combinations ofthese peptides.

FIG. 25. Graph showing number of surface metastases in mice bearingB16-F10-LacZ tumor cells and treated with combinations of peptides.

DETAILED DESCRIPTION

This disclosure provides four peptides:

peptide amino acid sequence SEQ ID NO: QP20 QTRTVPMPKIHHPPWQNVVP 1 HD20HHHQVYQVRSHWTGMHSGHD 2 WQ20 WNLPASFHNHHIRPHEHEWIQ 3 SQ20SSYHHFKMPELHFGKNTFHQ 4These peptides share a core sequence of HH_, which is shown above inbold, and have a strong affinity for the checkpoint receptor “programmeddeath 1” (PD-1). These peptides block the interaction of PD-1 with itsligand PD-L1 and can therefore be used to inhibit the progression of ahyperproliferative disorder, including cancer, or to treat infectiousdiseases, including persistent infections by agents such as HIV,hepatitis B virus (HBV), hepatitis C virus (HCV), and Plasmodiumfalciparum, by enhancing, stimulating, and/or increasing an individual'simmune response.

Pharmaceutical Compositions

Pharmaceutical compositions comprise up to four of the peptidesdisclosed herein and a pharmaceutically acceptable vehicle. The“pharmaceutically acceptable vehicle” may comprise one or moresubstances which do not affect the biological activity of the peptidesand, when administered to a patient, does not cause an adverse reaction.Pharmaceutical compositions may be liquid or may be lyophilized.Lyophilized compositions may be provided in a kit with a suitableliquid, typically water for injection (WFI) for use in reconstitutingthe composition. Pharmaceutical compositions can be administered by anysuitable route, including, but not limited to, intravenous,intramuscular, intradermal, intraperitoneal, and subcutaneousadministration.

In some embodiments, one or more of the disclosed peptides can beconjugated to various moieties, such as albumin and transthyretin, toenhance the peptide's plasma half-life. Methods of preparing suchconjugates are well known in the art (e.g., Penchala et al., 2015;Kontermann, 2016; Zorzi et al., 2017).

Therapeutic Uses

Pharmaceutical compositions disclosed herein have a number oftherapeutic applications. In some embodiments, a pharmaceuticalcomposition disclosed herein can be administered to a patient to inhibitthe progression of a hyperproliferative disorder, such as cancer. Suchinhibition may include, for example, reducing proliferation ofneoplastic or pre-neoplastic cells; destroying neoplastic orpre-neoplastic cells; and inhibiting metastasis or decreasing the sizeof a tumor.

Examples of cancers that can be treated using a pharmaceuticalcomposition disclosed herein include, but are not limited to, melanomas,lymphomas, sarcomas, and cancers of the colon, kidney, stomach, bladder,brain (e.g., gliomas, glioblastomas, astrocytomas, medulloblastomas),prostate, bladder, rectum, esophagus, pancreas, liver, lung, breast,uterus, cervix, ovary, blood (e.g., acute myeloid leukemia, acutelymphoid leukemia, chronic myeloid leukemia, chronic lymphocyticleukemia, Burkitt's lymphoma, EBV-induced B-cell lymphoma).

In some embodiments, a pharmaceutical composition disclosed herein canbe administered in conjunction with a cancer vaccine. A “cancer vaccine”is an immunogenic composition intended to elicit an immune responseagainst a particular antigen in patient to which the cancer vaccine isadministered. A cancer vaccine typically contains a tumor antigen whichis able to induce or stimulate an immune response against the tumorantigen. A “tumor antigen” is an antigen that is present on the surfaceof a target tumor. A tumor antigen may be a molecule which is notexpressed by a non-tumor cell or may be, for example, an altered versionof a molecule expressed by a non-tumor cell (e.g., a protein that ismisfolded, truncated, or otherwise mutated). “In conjunction with”includes administration of the pharmaceutical composition may betogether with, before, or after administration of the cancer vaccine.

In some embodiments, a pharmaceutical composition disclosed herein canbe administered in conjunction with a chimeric antigen receptor (CAR) Tcell therapy to treat cancers in order to increase the efficacy of suchtherapy.

In some embodiments, a pharmaceutical composition disclosed herein canbe administered to a patient to treat infectious diseases, includingchronic infections, caused, e.g., by viruses, fungi, bacteria, andprotozoa, and helminths.

Examples of viral agents include human immunodeficiency virus (HIV),Epstein Barr Virus (EBV), Herpes simplex (HSV, including HSV1 and HSV2),Human Papillomavirus (HPV). Varicella zoster (VSV) Cytomegalovirus(CMV), and hepatitis A, B, and C viruses.

Examples of fungal agents include Aspergillus, Candida, Coccidioides,Cryptococcus, and Histoplasma capsulatum.

Examples of bacterial agents include Streptococcal bacteria (e.g.,pyogenes, agalactiae, pneumoniae), Chlamydia pneumoniae, Listeriamonocvtogenes, and Mycobacterium tuberculosis.

Examples of protozoa include Sarcodina (e.g., Entamoeba), Mastigophora(e.g., Giardia), Ciliophora (e.g., Balantidium), and Sporozoa (e.g.,Plasmodium falciparum, Cryptosporidium).

Examples of helminths include Platyhelminths (e.g., trematodes,cestodes). Acanthocephalins, and Nematodes.

In some embodiments a pharmaceutical composition disclosed herein can beadministered as a vaccine adjuvant in conjunction with a vaccine toenhance a response to vaccination (e.g., by increasing effector T cellsand/or reducing T cell exhaustion). “In conjunction with” includesadministration of the pharmaceutical composition may be together with,before, or after administration of, the vaccine. The vaccine can be, forexample, an RNA vaccine (e.g., US 2016/0130345. US 2017/0182150), a DNAvaccine, a recombinant vector, a protein vaccine, or a peptide vaccine.Such vaccines can be delivered, for example, using virus-like particles,as is well known in the art.

In some embodiments a pharmaceutical composition disclosed herein can beadministered to treat sepsis.

Example 1. Peptide Library Screening

The TriCo-20™ (TRICO-20™) and TriCo-16™ (TRICO-16™) Phage DisplayPeptide Libraries (Creative Biolabs, 45-1 Ramsey Road, Shirley, N.Y.11967) were screened to identify binders of soluble recombinant humanPD-1 receptor. After the fourth round of panning, obvious enrichment forspecific binders was observed, and individual peptides were confirmed asweakly specific binders in a clonal phage ELISA. A fifth round ofpanning led to greater enrichment. Table 1 lists four peptides whichshowed strong specific binding in the clonal phage ELISA.

TABLE 1 Clonal Phase ELISA coated uncoated Clone signal signalpeptide sequence SEQ ID NO: QP20 0.851 0.446 QTRTVPMPKIHHPPWQNVVP 1 HD200.281 0.109 HHHQVYQVRSHWTGMHSGHD 2 WQ20 0.275 0.115WNLPASFHNHHIRPHEHEWIQ 3 SQ20 0.284 0.159 SSYHHFKMPELHFGKNTFHQ 4

Example 2. Competitive PD-1:PD-L1 Binding Inhibition Assay

Briefly, detection of cell surface PD-1 on Jurkat cells was accomplishedby incubating cells with the human PD-L1-Fc fusion protein, followed bydetection of the recombinant molecule with a fluorescently labeledanti-human Fc antibody. Flow cytometry was performed to detect bindingbetween PD-1 and the PD-L recombinant protein. Quantitative bindingmeasurement was then determined by mean fluorescence intensity (MFI).

Jurkat Cell-surface expression of PD1 and binding of PD-L1 to thesecells were verified as shown in FIGS. 1 and 2. The results are shown inFIGS. 3A-B, 4A-B, 5A-B, and 6A-B.

Example 3. Cell-Based Reporter Assay

A cell-based reporter assay was used to assess whether binding of thefour peptides identified above was sufficient to block the interactionwith PD-1 and its ligand PD-L1. The components of the assay include aJurkat T cell line that stably expresses human PD-1 and a luciferasereporter, a CHO cell line that stably expressed human PD-L1, and apositive control anti-PD-1 antibody that blocks the interaction of PD-1and PD-L1, resulting in a measurable effect in the assay. The luciferasereporter in the Jurkat T cell line is triggered by IL-1, NFAT, or NF-κBresponse elements in the promoter region. The Jurkat T cells arepre-treated with CD3 and immediately cryopreserved for use in the assay.Interaction of the Jurkat T cells with the PD-L1 expressing cell lineinhibits the intracellular mechanism by which the luciferase constructis activated, thereby preventing luciferase expression. A molecule thatbinds to either PD-1 on the Jurkat T cells or to PD-L1 on the CHO cellssufficiently to prevent their interaction permits the Jurkat T cells toproduce luciferase. CellTiter-Glo® (CELLTITER-GLO®, Promega) was used tomeasure luciferase expression.

The results of positive control assays using the anti-PD-1 controlantibody are shown in FIGS. 7A-B. These results demonstrate that thecontrol antibody restores luciferase expression in a dose-dependentmanner, with peak-fold inhibition of approximately 8 at an antibodyconcentration of 20 μM.

The results of assays of the peptides identified above are shown inFIGS. 8A-B. These results demonstrate that each of the four peptidesrestores luciferase expression in a dose-dependent manner, withpeak-fold inhibition of approximately 1.5 at a concentration ofapproximately 25 μM.

Example 4. Tetanus Toxoid Recall Assay Using Individual Peptides

Peptides 1-4 were tested in a human PBMC-based tetanus antigen recallassay. “Peptide CQ-22” was used as a negative control.

PBMCs were obtained from plasma of human donors and tested in vitro forrecall of tetanus toxoid. Suitable PBMCs were cryopreserved untilneeded, then thawed and cultured in a 96-wellplate. Tetanus toxoid wasadded to the cultures in the presence or absence of peptides 1-4, andthe production of cytokines and cell surface T cell activation markerswere examined.

The results of these assays are shown in FIGS. 9-15 and summarizedqualitatively in Table 2. In the table, “x” indicates no effect, “−”indicates a possible low effect, “+” indicates some effect, and “++”indicates a definite effect.

TABLE 2 peptide IL-2 IL-4 IL-6 IL-10 IL-17a IFNγ TNFα QP20 x − x x x x xHD20 − x ++ x ++ ++ ++ WQ20 − ++ ++ x ++ ++ ++ SQ20 + − ++ + ++ ++ +

The results demonstrated a trend towards modest enhancement of IL-6,IL-17α, IFNγ, and TNFα production at the highest concentrations ofpeptides. No significant enhancement of IL-2 production was detected.

Example 5. Tetanus Toxoid Recall Assay Using Combinations of Peptides

Combinations of peptides were tested in the antigen recall assaydescribed above, using a different PBMC donor and a different lot numberof tetanus toxoid. The results are shown in FIGS. 16, 17, 18, 19, 20,21, and 22. These results demonstrated that the combination of the fourpeptides combination of the four peptides QP20, HD20, WQ20, and SQ20result in increased IL-2 production and reduced IL-17a production.

The effect of peptides QP20, HD20, WQ20, and QP20 on the production ofIL-2 and IL-17a appears to be donor-specific, as shown in FIGS. 23A-Band 24A-B.

Example 6. BIACORE® Assays

BIACORE® assays were carried out using a BIACORE® T-200 at 25° C. Theassay and regeneration buffers contained 10 mM HEPES (pH 7.4), 150 mMNaCl, 3 mM EDTA, and 0.05% P20. The immobilization buffer was 10 mMsodium acetate, pH 5.0. The flow rate used for immobilizing the ligandwas 5 μl/min. The flow rate for kinetics analysis was 30 l/min.

Scouting.

12,000 response units (RU) of human and 6000 RU of mouse PD-1 receptorswere directly immobilized on flow cell 2 and flow cell 4 of the CM5 chipby amine coupling method (EDC/NHS). The un-occupied sites were blockedwith 1M ethanol amine. Scouting was performed at a single analyteconcentration of 25 μM to confirm yes/no binding. Flow cell 1 was keptblank and used for reference subtraction. Binding of analyte to theligand was monitored in real time.

Full Kinetics.

Based on the scouting results, full kinetics were performed byimmobilizing higher RU of the ligand to a new chip and analyteconcentration at 25 μM, followed by serial dilution to 12.5, 6.25,3.125, 1.562, 0.78 and 0 μM concentration or as indicated. Due to faston rate and off rate, KD was determined by steady state equilibriumkinetics.

Chi square (χ²) analysis was carried out between the actual sensorgramand a sensorgram generated from the BIANALYSIS® software (black line) todetermine the accuracy of the analysis. A χ² value within 1-2 isconsidered significant (accurate) and below 1 is highly significant(highly accurate). The results are summarized in Table 3.

TABLE 3 Ligand 10,000 RU Analyte Rmax (RU) KA(1/M) KD (M) Conc. (μM) χ²mouse PD-1 WQ-21 270 1.31 × 10³ 7.61 × 10⁻⁴ 0-25 0.0203 mouse PD-1 QP-2013.4 1.80 × 10⁴ 5.54 × 10⁻⁵ 0-25 0.0446 mouse PD-1 HD-20 76 4.25 × 10³2.35 × 10⁻⁴ 0-25 0.11 mouse PD-1 SQ-20 12.8 2.14 × 10⁴ 4.68 × 10⁻⁵ 0-250.039 human PD-1 WQ-21 84.7 3.28 × 10³ 3.05 × 10⁻⁴ 0-25 0.0309 humanPD-1 QP-20 3.83 9.36 × 10⁴ 1.07 × 10⁻⁵ 0-25 0.0569 human PD-1 HD-20 3.353.18 × 10⁵ 3.41 × 10⁻⁶  0-12.5 0.0733 human PD-1 SQ-20 4.05 1.94 × 10⁵5.16 × 10⁻⁶ 0-25 0.111 mouse PD-1 Mouse PD-L1 259 2.75 × 10⁶ 3.64 × 10⁻⁷0-50 0.105 human PD-1 Human PD-L1 213 6.92 × 10⁶ 1.44 × 10⁻⁷ 0-50 2.44

These results indicate that each of the four peptides bind both humanand mouse PD-1. QP20 and SQ20 showed the highest affinity towards mousePD-1. HD20 and SQ20 showed the highest affinity towards human PD-1.

Example 7. Experimental Metastasis Model

Efficacy of the peptides was evaluated in a B16-F10-LacZ experimentalmetastasis model. In this model, B16-F10-LacZ cells, transfected toexpress the LacZ gene that encodes β-galactoside, an intracellularenzyme, are injected into the tail vein of syngeneic mice. The cellstravel through the circulation, settle in the lungs, and form tumors.Mice are terminated 2 weeks after implant. When the enzyme cleaves itssubstrate, X-gal, the products dimerize and change color and can bedetected ex vivo. The number of metastatic tumors on the surface of thelung is then quantified by manual counting of tumors under a dissectingmicroscope.

Briefly, mice (N=7) were implanted on study day 0 with B16-F10-LacZtumor cells (5×10⁵ or 1×10⁶ cells per mouse) by intravenous injection inthe tail vein. Mice received a treatment of the peptide combination (200μg, 20 μg, or 2 μg, each peptide per dose) intravenously by tail veininjection on study days 2, 5, 7, 9 and 12. Detailed clinicalexaminations and body weights were recorded regularly during treatment.Mice were terminated on study day 14, and their lungs were removed andstained. The number of tumor metastases were counted. Treatment groupsare described in Table 4.

TABLE 4 Group N Implant Treatment Dose Route Treatment Days 1 7 5 × 10⁵QP-20, SQ-20, HD-20, WQ-20 200 μg IV SD 2, 5, 7, 9, 12 2 7 5 × 10⁵QP-20, SQ-20, HD-20, WQ-20  20 μg IV SD 2, 5, 7, 9, 12 3 7 5 × 10⁵QP-20, SQ-20, HD-20, WQ-20  2 μg IV SD 2, 5, 7, 9, 12 4 7 5 × 10⁵Untreated — — — 5 7 1 × 10⁶ QP-20, SQ-20, HD-20, WQ-20 200 μg IV SD 2,5, 7, 9, 12 6 7 1 × 10⁶ Untreated — — —

The results are shown in FIG. 25. A good dose response was observed whenmice were implanted at both cell concentrations. Mice treated with thehighest dose of peptide mixture (200 μg) had the fewest tumors (average97), and mice treated with the lowest dose of peptide mixture (2 μg) hadthe most tumors (average 205). Similarly, in the two groups that wereimplanted with high tumor numbers, the untreated group had significantlymore tumors. This indicates that the 4 peptides in combination showed adose-dependent efficacy on B16-F10-LacZ tumor growth in vivo. Moreover,the peptide combination was well tolerated by the mice and did not haveany acute adverse effects on animal health.

Example 8. Effect of Peptide Combination on the Immunogenicity of aMalaria Vaccine

Immunogenicity of the peptide combination as a prophylactic vaccineadjuvant was assessed in a mouse model of malaria. Balb/c mice immunizedwith an adenovirus-based malaria vaccine expressing the Plasmodiumyoelli circumsporozoite protein (AdPyCS) were given 200 μg of thepeptide combination, anti-PD-1 mAb, anti-PDL1 mAb, or the negativecontrol peptide ovalbumin (OVA) on days 1, 3, 5, and 7 afterimmunization with AdPyCS (Table 5). Note that no additional adjuvant wasadded to the AdPyCS antigen. Spleens were collected 12 days afterimmunization, and the number of splenic PyCS-specific, IFNγ-secretingCD8⁺ T cells was determined via ELISpot assay. Note that for the ELISpotassay, splenocytes were stimulated with the SYVPSAEQI peptide (SEQ IDNO:5), an H-2Kd-restricted CD8⁺ T cell epitope of PyCS.

TABLE 5 Treatment Cohort Test Sample # Mice Route days 1 AdPyCS only 5 —— 2 AdPyCS + control OVA peptide 5 i.p. 0, 1, 3, 5, 7 (200 μg) 3AdPyCS + peptide combo 5 i.p. 0, 1, 3, 5, 7 (200 μg) 4 AdPyCS +anti-PD-1 antibody 5 i.p. 0, 1, 3, 5, 7 (200 μg) 5 AdPyCS + anti-PDL1antibody 5 i.p. 0, 1, 3, 5, 7 (200 μg)

Significant differences in the average number±standard deviation ofCSP-specific, IFNγ-secreting CD8⁺ T cells per 0.5×10⁶ splenocytesbetween the AdPyCS alone (Cohort 1) and the peptide combination (Cohort3), anti-PD-1 antibody (Cohort 4) or anti-PD-L1 antibody (Cohort 5) weredetected using the one-way ANOVA test (*** p<0.001, and * p<0.05). Theseresults demonstrate that the peptide combination (Cohort 3) isfunctionally active in vivo, increasing the number of CSP-specific.IFNγ-secreting CD8⁺ T cells ˜1.6-fold relative to AdPvCS alone (Cohort1), which was similar to changes with anti-PD-1 or -PD-L1 antibody(Cohort 4 and 5).

Example 9. Effect of Peptide Combination on Survival in a Model ofSepsis

Sepsis can negatively alter T cell function and survival, however thiscan be reversed when the PD-1:PDL1 interaction is blocked, which resultsin improved survival. Thus the efficacy of the peptide combination wasassessed in a representative, clinically relevant model of sepsis whereCD1 mice are subjected to cecal ligation and puncture (CLP) to induceintra-abdominal peritonitis. For this study, 200 μg of either thepeptide combination or anti-PD-1 antibody were administered i.v. at 2,24, 48, 72 and 96 hours after surgery. A vehicle control group was alsoincluded. Six mice were in each group. All mice were checked twice dailyfor signs of morbidity and mortality. Administration of the peptidecombination conferred an enhanced survival advantage over the vehiclecontrol group where the peptide combination showed a 2-fold highersurvival rate (Table 6). Moreover, survival in the peptide combinationgroup was slightly above treatment with anti-PD-1 antibody.

TABLE 6 Group % Survival Vehicle Control 50% Anti-PD-1 antibody 83% PD-1Peptide Combo 100% 

Example 10. Effect of Peptide Combination on Serum HBsAg Levels inHBV-Infected Mice

The combination of QP20, HD20, WQ20, and SQ20 peptides was assessed in ahepatitis B virus (HBV) mouse model where the role of PD-1 in T cellexhaustion and immunotolerance is documented (Tzeng et al., 2012; Ye etal., 2015). PD-1 is elevated in the hepatic T cells of mice withpersistent HBV infection but not in animals that have cleared theinfection. In this model, it has been shown that inhibition of thePD-1/PD-L1 interaction with an anti-PD-1 mAb both increasesantigen-specific IFNγ production by hepatic T cells and reverses HBVpersistence (Tzeng et al., 2012). This mouse model of persistent HBVpresented an opportunity to test whether the combination of QP20, HD20,WQ20, and SQ20 peptides can reverse T cell exhaustion in vivo and aidthe immune system in controlling viral infection.

Mice infected with HBV were treated with saline (negative control), 200μg of QP20, HD20, WQ20, and SQ20 peptides combined, or 200 μg anti-PD-1mAb at 9 time points, 2 days prior to infection and days 1, 3, 6, 9, 12,14, 17 and 20 post infection. The level of serum HB surface antigen(HBsAg) was monitored by ELISA on days 7, 14, and 21 to follow theinfection (higher levels of serum HBsAg are reflective of higher viraltiter) and detect the immune enhancement activity of the combination ofQP20, HD20, WQ20, and SQ20 peptides. The group treated with thecombination of QP20, HD20, WQ20, and SQ20 peptides showed significantlylower mean level of serum HBsAg at weeks 2 and 3 post infection (p<0.05,1-way ANOVA, Tukey's Multiple Comparison Test) compared to the salinenegative control.

REFERENCES

-   Kontermann, “Half-life extended biotherapeutics,” Expert Opin. Biol.    Ther. 16, 903-15, 2016.-   Penchala et al., “A biomimetic approach for enhancing the in vivo    half-life of peptides,” Nat. Chem. Biol. 11, 793-98, 2015.-   Tzeng et al., “PD-1 blockage reverses immune dysfunction and    hepatitis B viral persistence in a mouse animal model,” PLoS One    7(6):e39179, 2012.-   Ye et al., “T-cell exhaustion in chronic hepatitis B infection:    current knowledge and clinical significance,” Cell Death Dis. March    19; 6:e1694, 2015.-   Zorzi et al., “Acylated heptapeptide binds albumin with high    affinity and application as tag furnishes long-acting peptides,”    Nature Communications 8, 16092, 2017.

The invention claimed is:
 1. A pharmaceutical composition, comprising:(a) up to four peptides selected from the group consisting of: (i) apeptide consisting of the amino acid sequence SEQ ID NO:1; (ii) apeptide consisting of the amino acid sequence SEQ ID NO:2; (iii) apeptide consisting of the amino acid sequence SEQ ID NO:3; and (iv) apeptide consisting of the amino acid sequence SEQ ID NO:4; and (b) apharmaceutically acceptable vehicle.
 2. The pharmaceutical compositionof claim 1, which comprises only one of the four peptides.
 3. Thepharmaceutical composition of claim 1, which comprises only two of thefour peptides, wherein the two peptides are selected from the groupconsisting of: (a) the peptide consisting of the amino acid sequence SEQID NO:1 and the peptide consisting of the amino acid sequence SEQ IDNO:2; (b) the peptide consisting of the amino acid sequence SEQ ID NO:1and the peptide consisting of the amino acid sequence SEQ ID NO:3; (c)the peptide consisting of the amino acid sequence SEQ ID NO:1 and thepeptide consisting of the amino acid sequence SEQ ID NO:4; (d) thepeptide consisting of the amino acid sequence SEQ ID NO:2 and thepeptide consisting of the amino acid sequence SEQ ID NO:3; (e) thepeptide consisting of the amino acid sequence SEQ ID NO:2 and thepeptide consisting of the amino acid sequence SEQ ID NO:4; and (f) thepeptide consisting of the amino acid sequence SEQ ID NO:3 and thepeptide consisting of the amino acid sequence SEQ ID NO:4.
 4. Thepharmaceutical composition of claim 1, which comprises only three of thefour peptides, wherein the three peptides are selected from the groupconsisting of: (a) the peptide consisting of the amino acid sequence SEQID NO:1, the peptide consisting of the amino acid sequence SEQ ID NO:2,and the peptide consisting of the amino acid sequence SEQ ID NO:3; (b)the peptide consisting of the amino acid sequence SEQ ID NO:1, thepeptide consisting of the amino acid sequence SEQ ID NO:2, and thepeptide consisting of the amino acid sequence SEQ ID NO:4; (c) thepeptide consisting of the amino acid sequence SEQ ID NO:2, the peptideconsisting of the amino acid sequence SEQ ID NO:3, and the peptideconsisting of the amino acid sequence SEQ ID NO:4; and (d) the peptideconsisting of the amino acid sequence SEQ ID NO:1, the peptideconsisting of the amino acid sequence SEQ ID NO:3, and the peptideconsisting of the amino acid sequence SEQ ID NO:4.
 5. The pharmaceuticalcomposition of claim 1, which comprises all four of the peptides.
 6. Thepharmaceutical composition of claim 2, wherein the one of the fourpeptides consists of the amino acid sequence SEQ ID NO:2.